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Animal procedures were carried out in compliance with Directive 2010/63/EU on the protection of animals used for experimental and other scientific purposes and approved by the Ethical Committee of the Institute for Biological Research ‘Siniša Stanković’, University of Belgrade, Serbia.
Male Dark Agouti (DA) and Albino Oxford (AO) rat strains (age 8–12 weeks) were used for the experiments. They were conventionally housed in a controlled environment (12-h light/dark cycle, 22 ± 2 °C, and 60% relative humidity) with free access to standard rodent chow and water in the unit for experimental animals at the Institute for Biological Research ‘Siniša Stanković’, Belgrade, Serbia.
In the prolonged Cd exposure experiments, the DA rats were exposed for 30 d to Cd doses that included the environmentally relevant Cd concentrations to which humans are exposed[52-54]. Cadmium chloride (CdCl2) was prepared in distilled water at concentrations of 5 mg/L (5 ppm) and 50 mg/L (50 ppm) of the Cd (II) ion. Control rats were given distilled water. Eight rats were assigned per group in two independent experiments.
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Prior to tissue collection, the animals were anesthetized with an intraperitoneal injection of 15 mg/kg body weight of Zoletil 100 (Virbac, Carros, France). The lungs were aseptically removed, cleared of blood, and finely minced. Lung leukocytes were isolated following incubation by gentle mixing for 30 min at 37 °C in RPMI-1640 culture medium supplemented with 2 mmol/L glutamine and 20 µg/mL gentamycin in the presence of 1 mg/mL collagenase type IV (Worthington Biochemical Corp., Lakewood, NJ, USA) and 30 µg/mL DNase I (Sigma Chemical Co., St. Louis, MO, USA). The cells were resuspended in culture medium supplemented with 5% (v/v) heat-inactivated fetal calf serum and voriconazole (5 µg/mL) (Pfizer PGM, Poce Sur Cisse, France). Total cell counts and viability were determined by trypan blue exclusion using a LUNA-IITM automated cell counter (Logos Biosystems, Anyang, South Korea).
Isolated leukocytes from healthy untreated animals (4 × 106 cells/well in a 96-well plate) were cultured for 48 h with various Cd concentrations (1, 5, 10, and 50 μmol/L) alone and in the presence of 3 µmol/L of the AhR antagonist CH-223191 (Sigma-Aldrich) to determine cytokine production and to isolate the RNA. The Cd doses used for cell stimulation were chosen to fit the most commonly used Cd doses in in vitro studies using cells from both human[21,44,45] and animal origins[29]. In a preliminary set of experiments, the cells were first pretreated for 1 h with CH-223191 and then Cd was added. No differences were noted between pretreated cell cultures and cells concomitantly treated with Cd and the antagonist, so the results obtained in cells co-treated with Cd and the antagonist are presented.
Leukocyte viability was measured by the MTT reduction assay following 48 h of culture. MTT (500 µg/mL) was added to each well of a 96-well plate and incubated for 3 h at 37 °C in a humidified atmosphere of 5% CO2. The formazan that formed was dissolved during an overnight incubation with 10% sodium dodecyl sulfate-0.01 N HCl, and absorbance of the extracted chromogen was read spectrophotometrically at 540 nm.
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Lung leukocytes (1 × 106) were collected after a 48 h treatment with Cd and lysed in 10 mmol/L HCl. After precipitating the protein with 5% sulfosalicylic acid, the GSH level was quantified in the supernatant using 5,5'-dithio-bis-[2-nitrobenzoic acid] (DTNB) in Tris-HCl (pH 8.9) and reduced glutathione as the standard[55] spectrophotometrically at 412 nm. The GSH level was expressed as μmol of GSH/mg protein.
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The dihydrorhodamine 123 assay (DHR 123; Life Technologies Corp. Carlsbad, CA, USA), based on the oxidation of DHR 123 to fluorescent rhodamine 123 by hydrogen peroxide, was used to measure ROS levels in lung leukocytes[56]. Lung leukocytes (1 × 106) treated with Cd for 48 h were incubated for 1 h in medium containing 4 μmol/L DHR 123. After the incubation, the cells were washed with PBS, fixed in 1% paraformaldehyde, and assayed for fluorescence intensity on the CyFLOW SPACE (Partec, Munich, Germany). A minimum of 10,000 events was acquired each time.
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Cytokine concentrations were determined in lung leukocyte-conditioned medium using commercially available ELISA kits for rat IL-1β and IL-6 (R&D Systems, Minneapolis, MN, USA), and TNF (eBioscience Inc., San Diego, CA, USA) according to the manufacturer’s instructions. Cytokine titers were calculated with reference to a standard curve constructed using known amounts of the respective recombinant cytokine standards provided in the kits. The results are presented as relative change compared to the control (Cd 0 µmol/L, considered 1).
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Lung leukocytes stimulated in vitro with Cd and leukocytes isolated from animals exposed for 30 d to Cd were used to isolate RNA. RNA (1 µg) was isolated using the mi-Total RNA Isolation Kit (Metabion, Martinsried, Germany) and reverse transcribed using random hexamer primers and MMLV (Moloney Murine Leukemia Virus) reverse transcriptase (Fermentas, Vilnius, Lithuania), following the manufacturer’s instructions. The prepared cDNAs were amplified using the Power SYBR® Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) based on the manufacturer’s recommendations in a total volume of 20 μL in a Quant StudioTM 3 Real-Time PCR Instrument (96-well, 0.2-mL) (Applied Biosystems). The thermocycler conditions were: hold stage at 50 °C for 2 min, followed by 95 °C for 10 min; the PCR stage at 95 °C for 15 s followed by 40 cycles of 60 °C for 60 s each; and a melt curve stage at 95 °C for 15 s, followed by 60 °C for 60 s and 95 °C for 1 s. The PCR primers (forward/reverse) used in this study are listed in Table 1. The PCR products were detected in real-time and the results were analyzed with Quant StudioTM Design & Analysis Software v1.4.3 (Applied Biosystems) and calculated as 2−ΔCt where ΔCt is the difference between the threshold cycle (Ct) values of a specific gene and the endogenous control (β-actin).
Item Forward Reverse β-actin (housekeeping reporter gene) 5'-CCCTGGCTCCTAGCACCAT-3' 5-'GAGCCACCAATCCACACAGA-3' CYP1A1 5'-GGGGAGGTTACTGGTTCTGG-3' 5'-CGGATGTGGCCCTTCTCAAA-3' CYP1B1 5'-CTCATCCTCTTTACCAGATACCCG-3' 5'-GACGTATGGTAAGTTGGGTTGGTC-3' IL-6 5'-GCCCTTCAGGAACAGCTATGA-3' 5'-TGTCAACAACATCAGTCCCAAG-3' TNF-α 5'-TCGAGTGACAAGCCCGTAGC-3' 5'-CTCAGCCACTCCAGCTGCTC-3' IL-1β 5'-CACCTCTCAAGCAGAGCA-3' 5'-GGGTTCCATGGTGAAGTCAAC-3' NLRP3 5'-CAGAAGCTGGGGTTGGTGAA-3' 5'-CCCATGTCTCCAAGGGCATT-3' AhRR 5'-CAGCAACATGGCTTCTTTCA-3' 5'-GAAGCACTGCATTCCAGACA-3' AhR 5'-GCTGTGATGCCAAAGGGCAGC-3' 5'-TGAAGCATGTCAGCGGCGTGGAT-3' Table 1. List of the primers used in the study
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The results were pooled from two independent experiments with four animals per group per experiment and presented as mean ± standard error. Data were analyzed by analysis of variance followed by Tukey’s test to examine differences between the groups. P-values < 0.05 were considered significant.
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The expression of CYP mRNAs was measured using a DA rat model of prolonged oral metal exposure[39, 41], in which increased levels of Cd are noted in the lungs[41]. The CYP1A1 mRNA remained unchanged (Figure 1A) and CYP1B1 mRNA level increased (Figure 1B) in lung leukocytes of animals treated with either the low (5 ppm) or high (50 ppm) Cd dose compared to the controls (Cd 0 ppm). In addition, an increase in the AhR mRNA level was documented in lung leukocytes of Cd-exposed animals (0.0441 ± 0.0022 in response to the low, and 0.0458 ± 0.0039 in response to the high Cd dose compared to 0.0218 ± 0.0036 in the controls, P < 0.05).
Figure 1. Expression of CYP1A1 (A) and CYP1B1 (B) mRNA in lung leukocytes isolated from animals orally exposed to Cd (5 and 50 ppm). Results are presented as mean ± standard error. Significance at: *P < 0.05 vs. control (Cd dose 0 ppm).
To verify these findings, we exposed lung leukocytes isolated from healthy untreated animals to increasing Cd concentrations in vitro. Cell viability following stimulation with Cd revealed that 50 µmol/L Cd resulted in the death of 15.6% of the cells in culture, so non-lethal doses (i.e., 1, 5, and 10 µmol/L) were tested further. Leukocytes responded to Cd exposure by increasing the expression of CYP1A1 mRNA at all doses tested (Figure 2A) and CYP1B1 at 1 and 5 µmol/L (Figure 2B).
Figure 2. Expression of CYP1A1 (A) and CYP1B1 (B) mRNA in lung leukocytes isolated from healthy untreated animals following in vitro Cd exposure (1, 5, and 10 µmol/L). Results are presented as mean ± standard error. Significance at: *P < 0.05, **P < 0.01, and ***P < 0.001 vs. control (Cd dose 0 µmol/L).
Exposure to Cd was not accompanied by oxidative stress as judged by the lack of changes in the DHR assay and intracellular GSH (Table 2).
Parameters tested Cadmium dose (µmol/L) 0 1 5 10 DHR (mean fluorescence intensity) 1.72 ± 0.12 1.63 ± 0.06 1.63 ± 0.04 1.65 ± 0.05 Intracellular GSH (µmol/L per mg protein) 795.0 ± 88.7 846.7 ± 62.9 762.9 ± 81.4 738.6 ± 67.7 Table 2. Oxidative stress in lung leukocytes following in vitro Cd stimulation
To determine if increased expression of CYPs in response to Cd is a consequence of AhR activation, we measured CYP1A1 and CYP1B1 mRNA levels in the presence of the AhR antagonist CH-223191. The presence of CH-223191 in the culture decreased CYP1A1 mRNA in cells treated with 0 and 1 µmol/L Cd and CYP1B1 mRNA at 1 and 5 µmol/L Cd (Figure 3). As the effect of the AhR inhibitor was observed at 1 and 5 µmol/L Cd, these Cd doses were used for the remaining experiments.
Figure 3. Expression of CYP1A1 (A) and CYP1B1 (B) mRNA in lung leukocytes co-treated with Cd and an AhR antagonist. Lines represent expression levels in cells treated with Cd alone (−CH-223191). Results are presented as mean ± standard error. Significance at: *P < 0.05 and ***P < 0.001 vs. control (Cd dose 0 µmol/L), #P < 0.05, and ###P < 0.01 for CH-223191 vs. −CH-223191.
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To determine if the effect of Cd on inflammatory cytokine production involves the AhR, IL-1β, IL-6, and TNF production and mRNA expression were measured in cells cultured with Cd alone and in the presence of the AhR antagonist CH-223191 (Figure 4). Lung leukocytes responded to Cd with increased IL-6 (Figure 4A) and decreased TNF (Figure 4B) and IL-1β (Figure 4C) production. The AhR antagonist diminished the effect of Cd and reversed cytokine production to the levels noted in cell cultures without Cd. Similarly, increased IL-6 (Figure 4D) and decreased TNF (Figure 4E) mRNA were noted in response to Cd stimulation. CH-223191 generally returned the mRNA levels to those comparable to the controls (except TNF where a higher mRNA level was noted in cells treated with 5 µmol/L Cd). However, in contrast to decreased production of IL-1β, mRNA of IL-1β increased following stimulation with Cd (Figure 4F). The AhR antagonist suppressed mRNA expression in cells treated with 1 µmol/L Cd, but had no effect on mRNA expression in response to 5 µmol/L Cd.
Figure 4. Cytokine production and gene expression by lung leukocytes isolated from healthy untreated animals following in vitro Cd exposure (1 and 5 µmol/L) in the absence or presence of CH-223191. IL-6 production (A) and mRNA expression (D). TNF production (B) and mRNA expression (E). IL-1β production (C) and mRNA expression (F). NLRP3 mRNA expression (G). Results are expressed as relative values compared to the control (considered 1, presented as a line on the graphs) and presented as mean ± standard error. Significance at: *P < 0.05, **P < 0.01 and ***P < 0.001 vs. control and #P < 0.05, ##P < 0.01, and ###P < 0.01 for CH-223191 vs.−CH-223191.
As activation of the AhR causes a decrease in IL-1β production by reducing the mRNA level of NLRP3, an inflammasome component[57], we next determined the NLRP3 level in cells treated with Cd (Figure 4G). As a result, the NLRP3 mRNA level decreased in response to Cd alone. Co-treatment of lung leukocytes with Cd and CH-223191 resulted in increased NLRP3 expression compared to the controls.
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Given that Cd toxicity is rat strain-dependent, the effect of Cd exposure on lung leukocytes from AO rats, a strain that is less sensitive to Cd immunotoxicity[58,59], was examined. In contrast to lung leukocytes from DA rats, CYP1A1 or CYP1B1 mRNA expression (Figure 5A) and proinflammatory cytokine production (Figure 5B) did not change in cells from the lungs of AO rats in response to Cd.
Figure 5. The effect of Cd on lung leukocytes isolated from healthy untreated AO rats. (A) Expression of CYP1A1 and CYP1B1 mRNA in lung leukocytes. (B) Relative cytokine production (IL-6, TNF, and IL-1 β) compared to the control (considered 1, presented as a line on the graphs). AhRR (C) and AhR (D) mRNA expression in lung leukocytes isolated from AO and DA rats. Results are presented as mean ± standard error. Significance at: *P < 0.05 and **P < 0.01 vs. control (Cd dose 0 µmol/L) and #P < 0.05, and ##P < 0.01 for AO vs. DA rats.
To determine what might have contributed to the lack of a Cd effect in AO rats, we measured the AhR repressor (AhRR) expression level, which is a natural regulator of the AhR, in Cd-treated leukocytes of both rat strains (Figure 5C). Generally higher levels of AhRR mRNA were noted in AO compared to DA rats. AhRR mRNA expression increased in DA rats at all Cd doses tested, but there were no changes in AO rats. The increased AhRR expression noted in DA rats may have been related to the increased AhR expression noted in leukocytes of this strain (Figure 5D).
Aryl Hydrocarbon Receptor is Involved in the Proinflammatory Cytokine Response to Cadmium
doi: 10.3967/bes2021.025
- Received Date: 2020-03-18
- Accepted Date: 2020-06-02
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Key words:
- Cadmium /
- Lung leukocytes /
- Aryl hydrocarbon receptor /
- Cytokine (IL-6, TNF, IL-1β) response
Abstract:
Citation: | KULAS Jelena, TUCOVIC Dina, ZELJKOVIC Milica, POPOVIC Dusanka, POPOV ALEKSANDROV Aleksandra, KATARANOVSKI Milena, MIRKOV Ivana. Aryl Hydrocarbon Receptor is Involved in the Proinflammatory Cytokine Response to Cadmium[J]. Biomedical and Environmental Sciences, 2021, 34(3): 192-202. doi: 10.3967/bes2021.025 |